ES2407679T3 - Selected nucleotide exchange enhanced with ANB modified oligonucleotides - Google Patents

Abstract

A procedure for the altered & selected of a double-stranded acceptor DNA sequence comprising combining the double-stranded acceptor DNA sequence with a donor oligonucleotide in the presence of proteins that are interchangeable with selected nucleotides, wherein the double-stranded DNA receptor sequence contains a first DNA sequence and a second DNA sequence that is the complement of the first DNA sequence and in which the donor oligonucleotide comprises a domain that comprises at least one discrepancy with respect to the double-stranded acceptor DNA sequence to be altered, preferably with respect to to the first DNA sequence, and in which the nucleatide with the discrepancy is modified, in which the oligonucleatide contains more than one DNA, in which each ANB is located at least one nucleotide away from the discrepancy, and wherein the oligonucleatide comprises a section containing two ANBs located symmetrically trically around the discrepancy at a nucleotide distance from the discrepancy, in which the procedure is not for the treatment of the human body or animal body by surgery or therapy, and in which the procedure is not for the selected alteration of DNA on human beings.

Description

Selected nucleolide exchange enhanced with ANB-modified oligonucleotides.

Field of the Invention

The present invention is about a procedure for the specific and selective allegation of a nucleolide sequence at a specific DNA site in a target cell by introducing an oligonucleotide into that cell. The result is the selected exchange of one or more nucleotides so that the sequence of the target DNA becomes that of the oligonucleotide when they are different. More particularly, the invention relates to the exchange of selected nucle6ides using modified oligonucleotides. The invention also relates to the application of the procedure.

Background of the invention

Genetic modification is the procedure of deliberate creation of changes in the genetic material of living cells with the purpose of modifying or more genetically encoded biological properties of that cell, or of the organism of which the cell is a part or in which it can regenerate. These changes may take the form of a deletion of parts of the genetic material, an addition of exogenous genetic material or changes in the existing nucleotide sequence of the genetic material. Procedures for the genetic modification of eukaryotic organisms have been known for more than 20 years and have found widespread application in plant, human and animal cells and in microorganisms for improvements in the fields of agriculture, human health, food quality and environmental protection Common genetic modification procedures consist of adding exogenous DNA fragments to the genome of a cell, which will then confer a new property on that cell or its organism above and above the properties encoded by existing genes (including applications in which the Expression of existing genes will be suppressed by it). Although many such examples are effective in obtaining the desired properties, these procedures, however, are not very precise, because there is no control over the genomic positions in which exogenous DNA fragments are inserted (and, therefore, , on the definitive levels of expression), and because the desired effect will have to be manifested on the natural properties encoded by the original and well balanced genome. In contrast, genetic modification procedures that result in the addition, deletion or conversion of nucleotides into predefined genomic loei will allow precise modification of existing genes.

The exchange of oligonucleotide-directed selected nucleotides (TNE, sometimes ODTNE) is a procedure based on the supply to the nucleus of the eukaryotic cell of synthetic oligonucleotides (molecules consisting of short stretches of nucleotide-like resins resembling DNA in its base pairing properties of Watson-Crick, but which may be chemically different from DNA) (Alexeev and Yoon, Nature Biotechnol. 16: 1343, 1998; Rice, Nature Biotechnol. 19: 321, 2001; Kmiec, J. Clin Inves. 112: 632, 2003). By deliberately designing a discrepant nucleotide in the oligonucleotide homolog sequence, the discrepant nucleotide can be incorporated into the DNA gene sequence. This procedure allows the conversion of isolated nucleotides or, at most, some nucleotides into existing loci, but it can be applied to create terminating codons in existing genes, resulting in an increase in their function,

or create changes in codons, resulting in genes encoding proteins with an altered amino acid composition (protein engineering).

The exchange of selected nucleotides (TNE) has been described in plant, animal and yeast cells. The first reports on TNE used what was called a chimera, which consisted of a self-complementary oligonucleotide that is designed to intercalate at the chromosomal target site. The chimera contains a discrepant nucleolide that forms the template to introduce the mutation into the chromosomal target. To select TNE events, most studies attempt to introduce a single nucleotide change into an endogenous gene that leads to herbicide resistance. The first examples using chimeras came from human cells (see the review: Rice et al., Na !. Biotech. 19: 321-326). The use of chimeras has also been successful in tobacco, rice and corn plant species (Beelham et al., 1999 Proc. Natl. Acad. Sc !. USA 96: 87748778; Kochevenko et al., 2003 Plant Phys. 132: 174-184 ; Okuzaki et al., 2004 Plant Cel! Rep. 22: 509-512). However, it was found that the activity of the chimeras was difficult to reproduce and, therefore, the activity of the TNE of single-stranded oligonucleotide (mc) TNEs has been tested. These have been found to give more reproducible results in wheat, yeast and human cells (Liu et al., 2002 Nuc. Acids Res. 30: 2742-2750; resena, parekh-Olmedo et al., 2005 Gene Therapy 12: 639- 646; Dong et al., 2006 Plant Cell Rep. 25: 457-65).

Several groups have shown that TNE can also be detected using cell total protein extracts. Such TNE activity search assays are called cell-free assays (Cole-Strauss et al., 1999 Nucleic Acids Res. 27: 1323-1330; Gamper et al., 2000 Nucleic Acids Res. 28, 4332-4339; Kmiec et al. , 2001 Plant J. 27: 267-274; Rice et al., 2001 40: 857-868). The test is configured as follows. A plasmid containing two bacterial antibiotic resistance genes (kanamycin and carbenicillin) is mutated so that one of the antibiotic resistance genes (for example, kanamycin) contains a terminator codon within the reading frame due to the a single nucleotide (for example, TAT to TAG). This mutated plasmid is then incubated with total cell protein and a monocalenary oligonucleotide designed to correct the terminator codon in the antibiotic resistance gene. The proteins needed for TNE are present in the cell extract and use the oligonucleotide to alter the terminating codon in the antibiotic resistance gene, restoring the resistance phenotype. The plasmidlco DNA is then purified from the reaction mixture and transformed into E. coli. Next, the bacteria are plated on media containing kanamycin, and the number of bacterial colonies represents the number of TNE repair events. The effectiveness of electroporation is calculated by counting the number of colonies that develop in media containing carbenicillin. The effectiveness of TNE can be quantified by calculating the proportion of plasmids repaired COn with respect to the total number of transformed plasmids.

In such an experiment, the oligonucleotide makes a substitution, altering TAG to TAC. In addition, the cell-free system can also be used to study the possibility of using oligonucleotides to produce individual nucleotide insertions. Plasmids can be produced that have a single nucleotide removed from the antibiotic resistance gene, generating a mutation of the reading frame. In the cell-free assay, the deletion is repaired by the addition of a nucleotide mediated by the 0ligonucleotide.

The biggest problem faced by the application of TNE in cells of higher organisms such as plants is the little efficacy that has been documented so far. In Malz, Zhu et al. (2000 Nature Biotech. 18: 555-558) documented a conversion frequency of 1 • 10 .... Further studies in tobacco (Kochevenko et al., 2003 Plant Phys. 132: 174- 184) and rice (Okuzaki et al., 2004 Plant Cell Rep. 22: 509-512) have documented frequencies of 1 • 10 "and 1 • 10" ", respectively. These frequencies are still too low for the practical application of TNE

Faithful DNA replication is one of the key criteria that mediates the maintenance of genomic stability and ensures that the genetic information contained in the DNA is transmitted free of mutation from one generation to the next. Many errors arise from the lesion to the parental DNA chain or are generated by agents that react with DNA bases (UV light, environmental toxins). Every body must maintain a safeguard to avoid or correct these mutations. It is believed that the discrepancy repair system (MRM) recognizes and controls discrepant or unpaired bases caused during DNA replication in the surveillance of DNA lesions and in the prevention of recombination between non-identical sequences (Fedier and Fink, 2004 Int. J Oncol. 2004; 24 (4): 1039-47), and contributes to the fidelity of DNA replication in living cells.

The TNE has been described in several Kmiec patent applications, inter alia in WOO173002, W003J027265, WOO1 / 87914, W099 / 58702, W097 / 48714, W002l10384. It is contemplated in WO 01/73002 that the low efficiency of gene aeration obtained using unmodified DNA oligonucleotides is generally believed to be a consequence of the degradation of the donor oligonucleotides by nucleases present in the reaction mixture or the target cell . To remedy this problem, it is proposed to incorporate modified nucleotides that make the resulting oligonucleotides resistant to nucleases. Typical examples include nucleotides with phosphorotloate bonds, 2'-0-methyl analogs or blocked nucleic acids (ANB). These modifications are preferably located at the ends of the oligonucleotide, leaving a central domain of DNA surrounding the selected base. In addition, the publication stipulates that specific chemical interactions are involved between the oligonucleotide converter and the proteins involved in the conversion. It is impossible to predict the effect of such chemical interactions to produce nuclease-resistant talT1lini using modifications other than the incorporation of ANB, phosphorothioate bonds or 2'-O-methyl analogs into the oligonucleotide, because the proteins involved in the aHeration process or its chemical interaction with the oligonucleotide substituents and, according to the inventors of W00173002, cannot be predicted.

Since the efficiency of the current ODTNE procedures is relatively low (as stated above, between 10 "and 10", despite documented high rates of oligonucleotide delivery of 90%), there is a need in the art to find procedures for TNE that are more effective. Consequently, the present inventors have proposed to improve the existing TNE technology.

Description of the invention

The present invention is disclosed in the claims. The present inventors have now discovered that by incorporating nucleotides into the donor oligonucleotide for TNE that are capable of binding more strongly to the acceptor DNA than the corresponding unmodified nucleotides such as A, C, T or G, the rate of TNE can be significantly increased. Without being restricted with theoretical considerations, the present inventors believe that by incorporating modified nucleotides into the donor oligonucleotide, the donor oligonucleotide binds more strongly to the acceptor DNA and, therefore, increases the proportion of TNE. The present inventors have discovered that oligonucleotides containing one or more AN B at positions close to the discrepancy, but not (directly) adjacent to it, that is, located at a distance of at least one nucleotide from the discrepancy, significantly improve the effectiveness of TNE.

To this end, the effect of the use of 0ligonucleotides incorporating one or more ANBs at various positions in the otigonucleotide on the frequency of TNE in the cell-free system has been investigated. The TNE activity of such oligonucleotides was compared with the TNE activity of oligonucleotides composed of normal DNA. It was found that oligonucleotides containing one or more ANB at positions at least one nucleotide away from the discrepancy increased the efficacy of TNE for both substitutions and insertions in the cell-free assay to a level not observed so far. Oligonucleotides containing ANB were up to 10 times more effective in the cell-free assay compared to oligonucleotides composed of normal DNA. It was also found that the efficiency of TNE could be further improved by increasing the number of ANBs in the oligonucleotide, whereby ANBs are located at a distance of at least 2, preferably at least 3, more preferably at least 4 nucleolides. In addition, it was found that the improvement observed was independent of lucus, indicating that the oligonucleotides of the invention with ANB in particular positions with respect to the discrepancy are capable of providing improved frequencies of TNE in a species independent manner, as in plant cells And animals.

The present invention is thus based on the inventive consideration that the exchange of selected selected nucleotides can be achieved through the use of partially modified oligonucleotides (i.e., at most 75%, preferably at most 50%) with ANB. The location, type and amount of oligonucleotide modification can be varied, within limits, as will be disclosed hereinbelow.

Thus, the present disclosure, in one aspect, provides oligonucleotides modified with ANB. MC oligonucleotides modified with ANB can be used to introduce specific genetic changes in plant, animal or human cells. The invention is applicable in the fields of biomedical research, agriculture and for building plants and animals, including humans, specifically mutated. The invention is also applicable in the field of medicine and gene therapy.

The sequence of an oligonucleotide of the invention is homologous to the target chain except for the part that contains a discrepant base that introduces the base change in the target chain. The discrepant base is introduced into the target sequence. Manipulating the modification (compared to conventional A, e, T or G) of the nucleotides and, more particularly, manipulating the location and amount of the oligonucleotide modification with ANB that introduces the discrepancy, can improve the efficiency (or degree of successful incorporation of the desired nucleotide at the desired position in the DNA doublet).

Another aspect of the disclosure lies in a procedure for the selected alignment of a parental DNA chain (first chain, second chain) by contacting the parental DNA doublet with an oligonucleotide containing at least one discrepant nucleotide compared to the parental chain , the donor oligonucleotide containing a section that is modified with ANB at particular positions so that it has a greater binding capacity than the parent chain (acceptor) in the presence of proteins that are capable of exchanging selected nucleotides.

Thus, the crux of the invention is found in the improvement in the linking capacity of the intercalating oligonucleotide (sometimes referred to as a donor) with ANB oligonucleotides with respect to the unmodified intercalating oligonucleotide, the modification being performed with ANB in one or more positions that are not They are adjacent to the discrepancy.

Detailed description of the invention

In one aspect, the disclosure refers to an oligonucleotide for the selected affixation of a double stranded DNA sequence containing a first DNA sequence and a second DNA sequence that is the complement of the first DNA sequence, the oligonucleotide comprising a domain. which is capable of hybridizing in the first DNA sequence, a domain that comprises at least one discrepancy with respect to the first DNA sequence, and the oligonucleotide comprising at least one section containing at least one modified nucleotide having a higher binding affinity that the A, e, T or G naturally present, the at least one nucleotide modified more strongly being linked with a nucleotide at an opposite position in the first DNA sequence, the at least one modified nucleotide being an ANB that is synchronized at a distance of at least one nucleotide with respect to the at least one discrepancy and, preferably, containing the ollgonucleotide, c At most, approximately 75% of modified nucleotides.

In one aspect, the disclosure refers to an ANB-modified ollgonucleotide for the selected alteration of a double stranded DNA sequence. The double stranded DNA sequence contains a first DNA sequence and a second DNA sequence. The second DNA sequence is the complement of the first DNA sequence and pairs with it forming a doublet. The oligonucleotide comprises a domain that comprises at least one discrepancy with respect to the double stranded DNA sequence to be altered. Preferably, the domain is the part of the oligonucleotide that is complementary to the first chain that includes the at least one discrepancy.

Preferably, the discrepancy in the domain is with respect to the first DNA sequence. The oligonucleotide comprises a section that is modified with at least one ANB to have a higher binding affinity than the (corresponding part of the) second DNA sequence. Preferably, the at least one modified nucleotide is an ANB that is located at a distance of at least one nucleotide from the at least one discrepancy; more preferably, the oligonucleotide contains, at most, approximately 75% of modified nucleotides.

The domain that contains the discrepancy and the section that contains the modified nucleotide (s) may be overlapping. Thus, in certain embodiments, the domain containing the discrepancy is located above the oligonucleotide at a different position in the section whose modification is considered. In certain embodiments. The domain incorporates the section. In certain embodiments. The section can enter the domain. In certain embodiments, the domain and section are located in the same position on the oligonucleotide and have the same length, that is, they coincide in length and position. In certain embodiments, there may be more than one section within a domain.

For the present invention, this means that the part of the oligonucleotide that contains the discrepancy to be incorporated in the DNA doublet can be placed in a different or displaced position from the part of the modified oligonucleotide. In particular, in certain embodiments in which the cell repair system (or, at least, the proteins involved in this system, or, at least, the proteins that are involved in the TNE) determines which of the chains contains the discrepancy and what chain should be used as a template for the correction of the discrepancy.

In certain embodiments, the oligonucleotide comprises a section containing at least one, preferably at least 2, more preferably at least three COn ANB modified nucleotides. In certain embodiments, the section on the oligonucleotide may contain more than 4, 5,6,7,8,9 or 10 AN6-modified nucleotides.

In certain embodiments, the at least one ANB is located at a distance of at most 10 nucleotides, preferably, at most, 8 nucleotides, more preferably, at most, 6 nucleotides, even more preferably, at most, 4, 3 or 2 nucle6tides with respect to the discrepancy. In a more preferred embodiment, the at least one ANB is located at a distance of 1 nucleotide with respect to the discrepancy; that is, a nucleotide is located between the discrepancy and the ANB. In certain embodiments relating to oligonucleotides containing more than one ANB, each ANB is located at a distance of at least one nucleotide with respect to the discrepancy. In a preferred embodiment, the ANBs are not located adjacent to each other, but separated from each other by at least one nucleotide, preferably two or three nucleotides. In certain embodiments, in the case of two or more (even numbers of) modifications with ANB of the oligonucleotide, the modifications are separated by a distance (approximately) equal to the discrepancy. In other words, preferably the modifications with ANB are located symmetrically in terms of discrepancy. For example, in a preferred embodiment, two ANBs are symmetrically located around the discrepancy at a distance of 1 nucleotide from the discrepancy (and 3 nucleotides from each other).

In certain embodiments, at most 50% of the modified oligonucleotide nucleotides are derived from ANB; that is, conventional A, C or G are replaced by their ANB counterpart, preferably at most 40%, more preferably at most 30%, even more preferably at most 20%, and most preferably, at most one 10%

In certain embodiments, more than one discrepancy may be introduced, either simultaneously or successively. The oligonucleotide can accommodate more than one discrepancy in locations either adjacent or separated in the oligonucleotide. To this end, the oligonucleotide may be adapted to accommodate a second set of ANBs that follow the principles outlined herein, with the proviso that they do not interfere with the greater mutual binding capacity due to the particular conformation of the ANBs in the oligonucleotide; that is, preferably separated around the discrepancy at a distance of 1 nucleotide with respect to the discrepancy. In certain embodiments, the oligonucleotide may comprise two, three, four or more discrepant nucleotides that may be adjacent or remote (ie, not adjacent). The oligonucleotide may further comprise additional domains and sections to accommodate this and, in particular, may comprise several sections. In certain embodiments, the oligonucleotide may incorporate a potential insert that is to be inserted into the acceptor chain. Such an insert may vary in length from more than five nucleotides to 100. Similarly, in certain embodiments, variations of similar lengths (from 1 to 100 nucleotides) can be introduced.

In a further aspect of the disclosure, the oligonucleotide design can be achieved:

determining the sequence of the chain will accept, or at least one section of the sequence by the nucleotide to be exchanged. Normally, this may be in the order of at least 10, preferably 15, 20, 25 or 30 nucleotides adjacent to the discrepancy, preferably on each side of the discrepancy (for example, GGGGGGXGGGGGG, X being the discrepancy);

designing a donor oligonucleotide that is complementary to one or both sections adjacent to the discrepancy and contains the desired nucleotide to be exchanged (for example "YCCCCCC);

providing (for example, by synthesis) the donor oligonucleotide with the modifications with ANB at the desired positions. Modifications can vary greatly, depending on the circumstances. Examples are CCCmCCmCYCCmCCCmC, CCCmCCCYCCCmCCC, CCCCCCYCCCmCmCmCm, CmCmCmCmCmCYCCCCCC, CCCCCmCYCCCmCCCCC, etc., representing Cm a nucleotide moiety modified with ANB. For a different acceptor sequence, for example ATGCGTACXGTCCATGAT, corresponding donor oligonucleotides can be designed, for example TACGCALGYCLGGTACTA (L = ANB), with a modification as variable as outlined hereinbefore;

by subjecting the DNA to be modified with the donor oligonucleotide in the presence of proteins that are capable of exchanging selected nucleotides, for example, and in particular, to proteins that are functional in the cellular mechanism for repairing discrepancies,

Without the restriction of theoretical considerations, it is believed that improved binding affinity increases the likelihood that an oligonucleotide will find its target and remain bound to it, improving the efficiency of TNE. Many different chemical modifications of the skeleton or sugar base confer an improved binding affinity. However, the present inventors decided to focus on ANB-modified oligonucleotides and found that their activity in TNE depends on the position in the oligonucleotide.

Blocked nucleic acid (ANB) is a DNA analog with very interesting properties for use in therapy with non-coding genes. ANBs are bicyclic and tricyclic nucleosides and nucleotide analogs and the 0ligonucleotides containing such analogs. The basic structures and functional characteristics of ANBs and related analogs are disclosed in various publications and patents, including WO 99114226, WO 00/56748, WOOO / 66604, WO 98139352, U.S. Patent No. 6,043,060 and U.S. Patent no

6,268,490.

Specifically, it combines the ability to discriminate between correct and incorrect targets (high specificity), with very high biostability (low rotation) and unprecedented affinity (very high binding force to the target). In fact, the affinity increase recorded with the ANB leaves the affinities of all previously documented analogs in the range of reduced to modest.

ANB is an analogue of RNA in which ribose is structurally limited by a methylene bridge between 2 'oxygen and 4' carbon atoms. This bridge restricts the flexibility of the ribofuranose ring and blocks the structure in a rigid bicyclic formation. This conformation, denominated of type N (or 3'-endo), results in an increase in the T m of the ANB that contains doublets and, consequently, greater affinities of bonds and greater specificities. Actually, NMR spectral studies have demonstrated the blocked N-type conformation of ANB sugar, but also revealed that ANB monomers are capable of twisting their unmodified neighboring nucleotides towards a N-type conformation. It is important that the characteristics ANB-favorable ones do not occur at the expense of other important properties, as is often observed in nucleic acid analogs.

The ANB can be freely mixed with all other chemical entities that make up the universe of DNA analogues. ANB bases can be incorporated into oligonucleotides of a length of only ANB sequences in their entirety or as long as ANB / DNA chimeras. ANBs can be placed in 3 'or 5' internal positions. However, due to their rigid bicyclic conformations, ANB residues sometimes alter the helical twisting of nucleic acid chains. Therefore, it is generally less preferable to design an oligonucleotide with two or more adjacent ANB residues. Preferably, the ANB residues are separated by at least one (modified) nucleotide that does not alter the helical twist, such as a conventional nucleotide (A, C, T or G).

The originally developed and preferred ANB monomer (the JB-D-oxi ANB monomer) has been modified creating new ANB monomers. The novel ANB a-L-oxy has superior stability against the activity of the 3 'exonuclease, and is also more potent and versatile than the JB-D-oxi ANB in the design of potent non-coding oligonucleotides. ANB xyl and ANB L-ribo can also be used, as disclosed in documents W09914226, WOOO / 56748, WOOOI66604. In the present invention any ANB of the above types is effective in achieving the goals of the invention, that is, greater efficiency of the TNE, with a preference for ANB analogs Jl-D.

In the TNE technique, the ANB modification has been included in a list of possible oligonucleotide modifications as an alternative to the qimeric molecules used in the TNE. However, there is no indication in the art so far that it suggests that ANB-modified single stranded DNA oligonucleotides significantly improve the efficiency of TNE to the point that is presently present when the ANB is located at a distance of at least one nucleotide. with respect to the discrepancy and / or the oligonucleotide does not contain more than about 75% (rounded to the nearest whole nucleotide) of ANB.

Oligonucleotide delivery can be achieved by electroporation or other conventional techniques that are capable of delivering either the nucleus O to the cytoplasm. An in vivo test of the present disclosure can be achieved using the cell-free system as described, indefinitely, in documents WOOl187914, W003 / 027265, W099 / 58702, W001 / 92512.

As used herein, the ability of the donor oligonucleotide to influence TNE depends on the type, location, and amount of modified nucleotides that are incorporated into the donor oligonucleotide. This capacity can be quantified, for example, by normalizing the binding affinity (or the binding energy (Gibbs free energy)) between conventional nucleotides at 1, that is, for both AT and GC bonds, the binding affinity is normalized in 1. For the oligonucleotides of the present invention, the relative binding affinity (RBA) of each modified nucleotide is> 1. This is exemplified in a formula below:

RBA = II RBA (modified) -II MB4 (not modified)> O,

n m

5 in which RBA is the relative affinity of total bond, RBA (modified) is the sum of the relative binding affinity of the modified oligonucleotide with a length of n nucle6tides and RBA (unmodified) is the sum of the relative affinity of bond of the unmodified oligonucleotide with a length of m nucleotides. For example, a 100 bp oligonucleotide contains 10 modifications, each with a relative binding affinity of 1.1. The total RBA is then equal to: RBA = [(10'1,1) + (90'1,0)] - (100'1,0) = 1.

10 Note that the definition of RBA is, in principle, independent of the length of the nucleotide chain being compared. However, when comparing RBAs of different chains, it is preferred that the chains be approximately the same length or that sections of comparable length be taken. Note that the RBA does not take into account that the modifications can be grouped together in a chain. Thus, a greater degree of modification of a certain A chain compared to a B chain means that R8A (A)> RBA (B). For the

15 sections comment both and comment below, corresponding RBA values can be defined and used. To accommodate the effect of the modified nucleotide position, a weighting factor can be entered in the RBA value. For example, the effect of a modified nucleotide on the donor oligonucleotide adjacent to the discrepancy may be greater than that of a modified nucleotide that is located at a distance separated five nucleotides from the discrepancy. In the context of this disclosure, RBA (donor »RBA (acceptor).

20 In certain embodiments, the donor's RBA value may be at least 0.1 greater than that of the acceptor's RBA. In certain embodiments, the donor's RBA value may be at least 0.2,0,3,0,4,0,5,0,6,0,7,0,8,0,9,1,0, 1.5, 2.0, 2.5 greater than the RBA of the acceptor. RBA values can be derived from conventional analysis of nucleotide binding affinity, such as by molecular modeling, thermodynamic measurements, etc. AKernatively, they can be determined by measuring differences of T m between modified and non-modified chains.

25 modified. As a matter of fact, the RBA can express as the difference in Tm between the unmodified and the modified chain, either by measurement or by calculation using conventional formulas for the calculation of the T m of a set of nucleotides, or by a combination of calculation and measurements.

The donor oligonucleotides according to the disclosure may contain additional modifications to improve hybridization characteristics, so that the donor has a greater affinity towards the target DNA chain 30 so that donor intercalation is easier. The donor oligonuCleotide can also be further modified so that it becomes more resistant to nucleases, to stabilize the triple or quadruple structure. Modification of ANB-modified donor oligonucleotides used in the invention may comprise phosphorothioate modification, 2-0Me substitutions, the use of additional ANB in the 3 and / or 5 'tennini of the oligonucleotide, APN (peptidonucleic acids), ribonucleotides and other bases that modify, preferably

35 improve the stability of the moisture between the oligonucleotide and the acceptor chain.

Particularly useful among such modifications are APNs, which are analogs of oligonucleotides in which the deoxybose skeleton of the oligonucleotide is replaced by a peptide skeleton. A peptide skeleton of such repeating units of N- (2-aminoethyl) glycine linked through amide bonds is constructed. Each peptide skeleton subunit is bound to a nucleobase (also called a "base '), which can be a naturally occurring base, not naturally present or modified. APN oligomers specifically link to a DNA sequence or Complementary affinity RNA with greater affinity than both DNA and RNA, consequently, the APN / DNA or APN / RNA resistant doublets have higher melting temperatures (Tm), and the Tm of APN / DNA or APNlRNA doublets is much less sensitive to salt concentration than DNA / DNA or DNA / RNA doublets The polyamide skeleton of APNs is also more resistant to enzymatic degradation.The synthesis of APNs is described, for example, in WO 92/20702 and WO 92/20703 Other APNs are illustrated, for example, in document W093 / 12129 and in US Patent No. 5,539,082, issued July 23, 1996. In addition, many scientific publications describe the synthesis of APNs, to Yes as its properties and uses. See, for example, Patel, Nature, 1993, 365, 490; Nielsen et al., Science, 1991, 254, 1497; Egholm, J. Am. Chem. Soc., 1992, 114, 1895; Knudson and others, Nucleic Acids Research,

50 1996, 24, 494; Nielsen et al., J. Am. Chem. Soc., 1996, 118, 2287; Egholm et al., Science, 1991,254, 1497; Egholm et al., J. Am. Chem. Soc., 1992, 114, 1895; And Egholm et al., J Am. Chem. Soc., 1992, 114,9677.

There are also additional useful modifications of the ANB oligonucleotides known as Super A and Super T, obtainable from Epoch Biosciences, Germany. These modified nucleotides contain an additional substituent that adheres to the main groove of the DNA, in which it is believed to improve the stacking of bases in the doublet of

55 DNA

In further embodiments, advantageous results can be obtained when, in addition to the 0B oligonucleotides modified with ANB, additional modifications are made to the oligonucleotide that further enhance the affinity of the 0ligonucleotide for the acceptor chain. Thus, it has been found that the ANB-modified 0ligonucleotide used in the present invention which, furthermore, comprises C5-modified pyrimidine and / or C7 -propyl modified slurries significantly improves the efficacy of TNE.

The donor 0ligonucleotides of the disclosure can also be created chimeric, that is, contain sections of DNA, RNA, ANB, APN or combinations thereof.

Thus, in certain embodiments, the oligonucleotide of the invention also contains other modified nucleotides, optionally not methylated.

In certain embodiments, the oligonucleotide is resistant to nucleases. This is advantageous in preventing the oligonucleotide from being degraded by nucleases and increases the likelihood that the donor oligonucleotide can find its target (acceptor molecule).

In certain embodiments of the disclosure, the nucleotide of the 0ligonucleotide at the position of the discrepancy may be modified. Whether or not the discrepancy can be changed will depend largely on the exact mechanism of the exchange of selected nucleotides or on whether the cellular DNA repair mechanism uses the difference in affinity between the donor and acceptor chains. The same applies to the exact location of the other modified positions in the immediate vicinity or neighborhood of the discrepancy. However, based on the disclosure presented herein, such an oligonucleolide can be designed and tested immediately, taking into account the test procedures for suitable oligonucleotides, as described elsewhere in this document. In certain embodiments, the nucleotide at the position of the discrepancy is not modified. In certain embodiments, the modification is in a position away from a nucleotide, preferably 2, 3, 4, 5, 6 or 7 nucleotides with respect to the discrepancy. In certain embodiments, the modification is placed in a position below the discrepancy. In certain embodiments, the modification is placed in a position both of the discrepancy. In certain embodiments, the modification is between 10 bp and 10 kb of the discrepancy, preferably between 50 and 5000 bp, more preferably between 100 and 500 bp of the discrepancy.

Oligonucleotides that are used as donors can vary in length, but generally vary in length between 10 and 500 nucleotides, with a preference for 11 to 100 nucleotides. preferably from 15 to 90, more preferably from 20 to 70, with 30 to 60 nucleids being most preferred.

In one aspect, the disclosure relates to a method for the selected alteration of a double-stranded acceptor DNA sequence comprising the combination of the double-stranded acceptor DNA sequence with a donor oligonucleotide, the double-stranded acceptor DNA sequence containing a first DNA sequence. and a second DNA sequence that is the complement of the first DNA sequence, and the donor oligonucleotide comprising a domain comprising at least one discrepancy with respect to the double-stranded acceptor DNA sequence to be altered, preferably with respect to the first DNA sequence, and a section of the donor oligonucleotide being modified with ANB to express a greater degree of affinity with the first DNA sequence compared to an unmodified nucleotide at that position of the oligonucleotide, in the presence of proteins that are capable of exchanging selected nucleotides, the ANB being located at a distance of a l minus one nucleotide with respect to the discrepancy.

The disclosure is, in its broadest form, generically applicable to all types of organisms such as humans, animals, plants, fish, reptiles, insects, fungi, bacteria, and so on. The disclosure is applicable for the modification of any type of DNA, such as DNA derived from genomic DNA, linear DNA, artificial chromosomes, nuclear chromosomal DNA, organelles chromosomal DNA, bacterial artificial chromosomes and artificial yeast chromosomes. The disclosure can be made in vivo, as well as ex vivo.

The disclosure is, in its broadest form, applicable for many purposes to alter a cell, correct a mutation by restoration to the natural form, induce a mutation, deactivate an enzyme by altering the coding region, modify the bioactivity of an enzyme altering the coding region, modifying a protein by altering the coding region.

The disclosure also deals with the use of 0ligonucleotides, essentially as described hereinbefore, stop a cell, correct a mutation by restoring to the natural form, induce a mutation, deactivate an enzyme by altering the coding region, modifying the bioactivity of an enzyme by altering the coding region, modifying a protein by altering the coding region, repair of discrepancies, selected alteration of genetic (plant) material, including gene mutation, repair of selected genes and gene blocking.

The disclosure also relates to kits, which comprise one or more oligonucleotides, as defined elsewhere in this document, optionally in combination with proteins that are capable of inducing MRM and, in particular, that are capable of TNE.

The disclosure is also about modified genetic material obtained by means of the process of the present disclosure, about cells and organisms that comprise the modified genetic material, and about plants or parts of plants that are obtained therein.

The disclosure relates in particular to the use of the TNE method using the modified oligonucleotides.

5 with ANB, as disclosed herein, to provide resistance to herbicides in plants. Plants that have been endowed with resistance to herbicides, in particular glyphosate and / or sulfonylurea herbicides, such as chlororsulfuron, are also disclosed.

Description of the figures

Figure 1: Schematic representation of the exchange of selected nucleotides. One gets in touch

A double stranded acceptor DNA chain containing a nucleotide (X) to be exchanged with an ANB modified donor oligonucleotide (schematically given as NNNmNNNmYNNmNNm) containing the nucleotide (V) to be inserted. The triple structure is subjected to an entome, or brought into contact therewith, that is capable of TNE or, at least, with proteins that are capable of carrying out TNE, such as those known as the enzyme free mixture of cells or a cell-free extract (see, inter afia, documents W099 / 58702,

15 WOO11730 (2).

Figure 2: Chemical structures of various ANB.

Figure 3: The efficiency of TNE repair of oligonucleotides containing ANB-modified nucleotides

20 as measured using the cell-free assay. For each experiment the repair efficiency was determined using the normal DNA oligonucleotide and set to a value of 1. The number of increases indicates the increase in repair seen using oligonucleotides containing ANB Il-D compared to the repair efficiency. obtained when the normal DNA oligonucleotide is used.

Example

25 Materials and procedures

Oligonucleotides containing ANB were purchased from Eurogentec. The sequences of the oligos used are shown below. The plasmid used in the experiments was a derivative of pCR2.1 (Invitrogen) that contains genes that confer resistance to both kanamycin and carbenicillin. End codons and deletions were introduced within the reading frame in the ORF of kanamycin and carbenicillin, as described

30 previously described (Sawano et al., 2000 Nucleic Acids Res. 28: e78). The KmY22stop plasmid has a TAT to TAG mutation in codon Y22 in the kanamycin ORF. In the KmY22 plasmid, the third nucleotide of codon Y22 (TAT) was deleted, giving a shift in the reading frame.

Kanamycin defective genes and oligonucleotides used in the cell-free system:

Km WT GAG AGG CTA TTC GGC TAT GAC TGG GCA CAA CAG

35 E R L F G Y D W

KrnY22stop GAG AGG CTA TTC GGC TAG GAC TGG GCA CM CAG

AND

R L F G *

KrnY22L1

GAG AGG CTA TTC GGC TA GAC TGG GCA CM CAG

AND

R L F G *

Oligo

Sequence ANB / DNA IDSEC

L1

tgtgcccagtCgTagccgaatagc 2/24 (8.3%) one

L2

tgtgcccagTcgtAgccgaatagc 2/24 (8.3%) 2

L3

TgTgCcCaGtCgTaGcCgAaTaGc 12/24 (50%) 3

L4

tGtGcCcAgTcGtAgCcGaAtAgC 12/24 (50%) 4

L5

tGtgcCcagTcgtAgccGaatAgc 6/24 (25%) 5

LB

tgtgCccagTcgtaGccgaAtagc 4/24 (16.6%)

40 The relevant sequence of the open reading frames of kanamycin and encoded amino acids are shown. Single nucleotide mutations that produce a terminator codon (TAG, *) as described above (Sawano et al., 2000 Nucleic Acids Res. 28: e78) were introduced. The 9 are shown in capital letters

ANB oligonucleotide sequences used in the experiments. The L1-L6 oligonucleotides were used to convert the KmY22stop and KmY22t mutation. in an alternative codon tyrosine coding (TAC). The discrepant nucleotide in each oligonucleotide is underlined. The oligonucleotide binding regions in the kanamycin ORF are underlined. The oligonucleotides L 1-L6 are complementary to the coding sequence of kanamycin.

Cell free assays were carried out as follows. Flower buds of Arabidopsis thaliana (ecotype Col.j) were collected and ground in a nitrogen atmosphere. 200 ~ I of protein isolation buffer (20 mM HEPES pH 7.5, 5 mM KCI, 1.5 mM MgCI2, 10mM DTT, 10% (v / v) glycerol, 1% (wlv) PVP) were added. The plant residues were pelleted by centrifugation at 14k RPM for 30 minutes and the supernatant was stored at -SO · C. Protein concentration was measured using the NanoOrange kit (Molecular Probes, inc.). A typical isolation resulted in a protein concentration of approximately 3-4 ~ gI ~ l. The cell-free reactions contained the following components: 1 ~ g plasmid DNA (KmY22stop or KmY22t.), 100 ng of oligonucleotide, 30 ~ g of total vegetable protein, 4 ~ I of sheared salmon sperm DNA (3 ~ g / ~ I), 2 ~ I of protease inhibitor mixture (conc. 50 ', Complete tablets of EDTA-free prolease inhibitor cocktail, Roche Diagnostics), 50 ~ I of cell-free reaction buffer 2. (400 mM Tris pH 7.5, 200 mM MgCI2, 2 mM DTT, 0.4mM spermidine, 50 mM ATP, 2 mM each of CTP, GTP, UTP, 0.1 mm each of dNTP and 10 mM NAD) compensated with water up to a total volume of 100 ~ I. The mixture was incubated at 37 ° C for 1 hour. Then the plasmid DNA was isolated as follows. 1 00 ~ I of H20 was added to each reaction to increase the volume, followed by 200 ~ I of phenal with alkaline buffer (pH 6-10). This was briefly subjected to vortexing and then centrifuged at 13k rpm for 3 minutes. The upper aqueous phase was transferred to a new tube and then 200 ~ I of chloroform were added. This was briefly subjected to vortexing, centrifuged at 13k rpm for 3 minutes and the aqueous phase was transferred to a new tube. The DNA was precipitated by the addition of 0.7 volumes of 2-propanol and the bead was resuspended in TE. To remove any copolymerized oligonucleotide, the DNA was passed through an Oiagen PCR purification column and the plasmid DNA was eluted in a final volume of 30 ~ I. 2 ~ 1 of plasmid DNA were electroporated into 18 ~ I of electrocompetent DH10B cells (invitrogen). After electroporation, the cells were allowed to recover in an SOC medium for 1 hour at 37 ° C. After this period, kanamycin was added to a concentration of 100 g / ml and the cells were incubated for an additional 3 hours. The solid medium contained 100 g / ml of kanamycin or carbenicillin. For the experiments with KmY22stop and KmY22, the number of TNE events on the kanamycin medium was detected and the electroporation efficiency was calculated by counting the number of colonies obtained from a 10 '"Y1 O" solution of the arranged electroporaclon in plates on the carbenicillin medium. The efficacy of TNE was calculated by dividing the number of TNE events by the total number of transformed cells.

Results

The oligonucleotides were designed to produce a single nucleotide substitution (KmY22stop) or insertion (KmY22A) into the terminator codon (TAG) introduced into the kanamycin ORF so that the codon re-encodes the correct amino acid.

In each experiment, unmodified DNA oligonucleotides and ANB modified oligonucleotides were used in parallel. In each experiment, the efficacy of the TNE obtained using the normal DNA oligonucleotide was arbitrarily set to 1, and the TNE efficacy of the ANB modified oligonucleotides was subsequently expressed as a number of increases with respect to the normal DNA oligonucleotide. It was found that the increase in repair efficiency depended on the position of the ANBs in the oligonucleotide. The oligonucleotide L 1 contained nucleotides flanking the discrepant nucleotide, and this showed no improvement with respect to a DNA oligonucleotide. However, oligonucleotide L2, with ANB nucleotides

separated from the discrepancy by an additional nucleotide, they presented an average increase of 5 times. The adition of

Extra ANB nucleotides (L3 and L4) decrease repair efficiency below that of a normal DNA oligonucleotide to the background level, and these oligonucleotides are biologically inactive. This is confirmed by the data using L5 and L6. In L5, in addition to the ANBs that flank the discrepant nucleotides as in L2, it also contains additional ANB nucleotides. In this case, the increase in repair obtained using L2 is not observed, presumably due to the inhibitory effects of additional ANB nucleotides. L6 also shows improvement in repair when using KmY22stop. The same trend is observed when experiments are carried out using KmY22t. It is known that repair by insertions of single nucleotides is less effective than repair by substitutions, and this is also what is observed here. L2 presents an increase in the repair frequency, which shows that, also for insertions, the position of the ANB nucleotides around the discrepancy is an important characteristic for increasing the repair frequency.

In the present disclosure, the experiments demonstrate that, using an in vitro TNE assay, the cell-free system, oligonucleotides containing ANB nucleotides show higher levels of TNE compared to the efficacy of TNE obtained using normal DNA oligonucleotides. This improvement can be up to 5 times. The effect is largely dependent on the position of the ANB nucleotides with respect to the discrepant nucleotide. In addition, the repair process is very sensitive to the number of ANB nucleotides in the oligonucleotide: when it reaches 50%, the oligonucleotide expresses a biologically reduced activity in the assay. Another form of ANB nucleotides, the ANB 2'-amino analogs (Rosenbohm et al., (2003) Org Biomol Chem. 1, 655-663), can be further functionalized by the addition of other chemical groups to the nucleotide. In addition to the higher binding affinity, such ANB nucleotides have additional chemical groups that can interact with DNA in many ways and that further increase binding affinity (Sorensen et al., (2003) Chem Commun (Camb) 17,2130- 2131). Such ANB 2'-amino can further increase the repair above the 5-fold increase already shown.

SEQUENCE LIST

<110> Keygene NV

<120> Exchange of selected nucle6tides enhanced with ANB-modified oligonucleotides

<130> P27711PC03

<150> PCT / NL20061OO0244

<151> 2006-05-09

<150> PCT INL2005J000884

<151> 2005-12-22

<160> 6

<170> Patentl n version 3.3

<210> 1 <211> 24

<212> DNA

<213> artificial

<220>

<223> TNB oligonucleotide modified with ANB

<220>

<221> modified base

<222> (11). :( 11)

<223> ANB-Cytosine

<220>

<221> modified_base

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<223> ANB-Tlrosina

<400> 1 tgtgcccagt cgtagccgaa tagc 24

<210> 2 <211> 24

<212> DNA

<213> artificial

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<223> TNB-modified TNE oligonucleotide

<220>

<221> modified_base

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<223> ANB-Tyrosine

<220>

<221> modified base

<222> (14). :( 14)

<223> ANB-Adenine

<400> 2 tgtgcccagt cgtagccgaa tagc 24

<210> 3 <211> 24

<212> DNA

<213> artificial

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<223> TNB oligonucleotide modified with ANB

<220> <221> modified_base

<222> (1) .. (1)

<223> ANB-Tyrosine

<220>

<221> modified_base

<222> (3) .. (3)

<223> ANB-Tyrosine

<220>

<221> modified_base

<222> (5) .. (5)

<223> ANB-Cytosine

<220>

<221> modified_base

<222> (7) .. (7)

<223> ANB-Cytosine

<220>

<221> modified_base

<222> (9) .. (9)

<223> ANB-Guanina

<220>

<221> modified_base

<222> (11) .. (11)

<223> ANB-Cytosine

<220>

<221> modified_base

<222> (13) .. (13)

<223> ANB-Tyrosine

<220>

<221> modified_base

<222> (15) .. (15)

<223> ANB-Guanina

<220>

<221> modified_base

<222> (17) .. (17)

<223> ANB-Cytosine

<220>

<221> modified_base

<222> (19) .. (19)

<223> ANB-Adenine

<220>

<221> modified_base

<222> (21) .. (21)

<223> ANB-Tyrosine

<220>

<221> modified_base

<222> (23) .. (23)

<223> ANB-Guanina

<400> 3 tgtgcccagt cgtagccgaa tage 24

<210> 4 <211> 24

<212> DNA

<213> artificial

<220>

<223> TNB-modified TNE oligonucleotide

<220>

<221> modified_base

<222> (2) .. (2)

<223> ANB-Guanina

<220>

<221> modified_base

<222> (4) .. (4)

<223> ANB-Guanina

<220>

<221> modified_base

<222> (6) .. (6)

<223> ANB-Cytosine

<220>

<221> modified_base

<222> (8) .. (8)

<223> ANB-Adenine

<220>

<221> modified base

<222> (10). :( 10)

<223> ANB-Tyrosine

<220>

<221> modified_base

<222> (12) .. (12)

<223> ANB-Guanina

<220>

<221> modified_base

<222> (14) .. (14)

<223> ANB-Adenine

<220>

<221> modified_base

<222> (16) .. (18)

<223> ANB-Cytosine

<220>

<221> modified_base

<222> (18) .. (18)

<223> ANB-Guanina

<220>

<221> modified_base

<222> (20) .. (20)

<223> ANB-Adenine

<220>

<221> modified_base

<222> (22) .. (22)

<223> ANB-Adenine

<220>

<221> modified_base

<222> (24) .. (24)

<223> ANB-Cytosine

<400> 4 tgtgcccagt cgtagccgaa tagc 24

<210> 5

<211> 24

<212> DNA

<213> artificial

<220>

<223> TNB Oligonucteotide Modified with ANB

<220>

<221> modified_base

<222> (2) .. (2)

<223> ANB-Guanina <220>

<221> modified_base

<222> (6) .. (6)

<223> ANB-Cytosine

<220>

<221> modified_base

<222> (10) .. (10)

<223> ANB-Tyrosine

<220>

<221> modified_base

<222> (14) .. (14)

<223> ANB-Adenine

<220>

<221> modified_base

<222> (18) .. (18)

<223> ANB-Guanina

<220>

<221> modified_base

<222> (22) .. (22)

<223> ANB-Adenine

<400> 5 tgtgcccagt cgtagccgaa tagc 24

<210> 6 <211> 24

<212> DNA

<213> artificial

<220>

<223> TNB oligonucleotide modified with ANB

<220>

<221> modified_base

<222> (5) .. (5)

<223> ANB-Cytosine

<220>

<221> modified base

<222> (10). :( 10)

<223> ANB-Tyrosine

<220>

<221> modified_base

<222> (15) .. (15)

<223> ANB-Guanina

<220>

<221> modified_base

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<400> 6 tgtgcccagt cgtagccgaa tagc 24

Claims (12)

one.

A method for the selected aeration of a double stranded acceptor DNA sequence comprising combining the double stranded acceptor DNA sequence with a donor oligonucleotide in the presence of proteins that are susceptible to exchange of selected nucleotides, in which the DNA sequence double stranded acceptor contains a first DNA sequence and a second DNA sequence that is the complement of the first DNA sequence and in which the donor oligonucleotide comprises a domain comprising at least one discrepancy with respect to the double stranded acceptor DNA sequence that has to be altered, preferably with respect to the first DNA sequence, and in which the nucleotide in the discrepancy is not modified, in which the oligonucleotide contains more than one ANB, in which each ANB is located at a distance of at least one nucleotide with respect to the discrepancy, and in which the oligonucleotide comprises a section containing two ANBs located symmetrically around the discrepancy at a distance of one nucleotide from the discrepancy, in which the procedure is not for the treatment of the human body or the animal body by surgery or therapy, and in which the procedure is not for the selected alteration of DNA in humans.

2.

The method according to claim 1 wherein at least 50% of the nucleotides of the oligonucleotide are derived from ANB.

3.

The method according to claim 1 wherein, in oligonucleotide 2, and no more, nucleotides are ANB.

Four.

The method according to any one of the preceding claims wherein the oligonucleotide is between 10 and 500 nucleotides in length.

5.

The method according to any one of the preceding claims wherein the alteration is within a cell preferably selected from the group consisting of a vagetal cell, a fungal cell, a rodent cell, a primate cell, a human cell or a cell of yeast.

6.

The method according to any one of claims 1-4 wherein the proteins are derived from a cell extract.

7.

The method according to claim 6, wherein the cell extract is selected from the group consisting of a plant cell extract, a fungal cell extract, a rodent cell extract, a primate cell extract, a human cell extract or an extract of yeast cells.

8.

The method according to any one of claims 1-4 wherein the alteration is a deletion, a substitution or an insertion of at least one nucleotide.

9.

The method according to any one of the preceding claims wherein the target DNA is derived from fungi, bacteria, plants, mammals or humans.

10.

The method according to any one of the preceding claims wherein the double-stranded DNA is derived from genomic DNA, linear DNA, mammalian artificial chromosomes, bacterial artificial chromosomes, artificial yeast chromosomes, artificial vagetal chromosomes, nuclear chromosomal DNA, organelles chromosomal DNA or Episomatic DNA

eleven.

The method according to any one of the preceding claims for airing a cell, correcting a mutation by restoration to the natural form, inducing a mutation, deactivating an enzyme by altering the coding ragion, modifying the bioactivity of an enzyme by altering the region coding or modifying a protein by altering the coding region.

12.

The use of an oligonucleotide as described in any one of claims 1 -4, to add a cell, correct a mutation by restoring to the natural form, induce a mutation, deactivate an enzyme by altering the coding ration, modify the bioactivity of an enzyme by altering the coding region, modify a protein by altering the coding region, repair discrepancies, the selected alteration of genetic (plant) material, including gene mutation, repair of selected genes and gene blockage, in which the Use is not for the treatment of the human body or animal body by surgery or therapy, and in which the use is not for the selected alteration of DNA in humans.

 11 111 111111 11 1) \ 1111111111 and  Fig 2   or.. ~ or" bu ANB p-D-oxi ANB P-O-uncle ANB a-L-oxi Fig 3